(1) Field of the Invention
This patent concerns inventions which improve the production of a class of material called polyhydroxyalkanoates (PHA) in higher plants. PHA is a group of bacterial polymeric material composed of linear polyesters of hydroxy acids and has thermoplastic properties. A low level production of PHA has previously been demonstrated in Arabidopsis thaliana transformed with the bacterial genes involved in PHA synthesis as described in Ser. No. 07/732,243, filed Jul. 19, 1991. In order to produce large amounts of PHAs in higher plants, the enzymes for PHA production have to be located in a subcellular compartment possessing a high level of precursor for PHA synthesis which is the plastid. The three genes of Alcaligenes eutrophus involved in synthesis of PHA from acetyl-CoA were modified to target the corresponding enzymes to the plastid of Arabidopsis plant cells as a specific example of the present invention.
(2) Description of Related Art
Polyhydroxyalkanoates (PHA), polyesters of 3-hydroxyacids, are produced as carbon storage reserves by a large variety of bacteria (Anderson, A. J. and Dawes, E. A., Microbiol. Rev. 54: 450-472, 1990). Poly-D-(-)-3-hydroxybutyrate (PHB), the most widespread and thoroughly characterized PHA, is a biodegradable and biocompatible thermoplastic.
Research on PHA production has been mainly concentrated on Alcaligenes eutrophus which produces short chain PHAs (C.sub.3 to C.sub.5 units). In A. eutrophus, PHB is synthesized from acetyl-CoA by the sequential action of three enzymes (FIG. 1) (Steinb uchel, A. and Schlegel, H. G., Mol. Microbiol. 5: 535-542, 1991). The first enzyme of the pathway, 3-ketothiolase (E. C. 2.3.1.9), catalyzes the reversible condensation of two acetyl-CoA moieties to form acetoacetyl-CoA. Acetoacetyl-CoA reductase (E.C. 1.1.1.36) subsequently reduces acetoacetyl-CoA to D-(-)-3-hydroxybutyryl-CoA, which is then polymerized by the action of PHB synthase to form PHB. PHB is produced as a polymer of 10.sup.5 -10.sup.6 monomer units which is accumulated in granules of 0.2 to 0.5 .mu.m in diameter, each granule containing approximately 1000 polymer chains (Anderson, A. J. and Dawes, E. A., Microbiol. Rev. 54: 450-472, 1990). When grown in medium containing glucose, A. eutrophus typically accumulates PHB up to 80% dry weight. The genes encoding the three phb biosynthetic enzymes described above have been cloned from A. eutrophus (Peoples, O. P. and Sinskey, A. J., J. Biol. Chem. 264: 15293-15297, 1989 and J. Biol. Chem. 264: 15298-15303, 1989; Slater, S. C., Voige W. H. and Dennis, D. E., J. Bacteriol. 170: 4431-4436, 1988, Schubert, P., Steinb uchel, A. and Schlegel, H. G., J. Bacteriol. 170: 5837-5847, 1988).
In addition to PHB homopolymer, A. eutrophus and other bacterial species can produce polymers containing various ratios of a number of different C.sub.3 to C.sub.5 monomers. The nature and proportion of these monomers is influenced by the carbon source supplied in the growth media. For example, when propionic acid or pentanoic acid is supplied to the fermentation feedstock, a random copolymer containing both 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) monomers is produced with a maximum of 43 mol % to 90 mol % of 3HV unit, respectively (Anderson, A. J. and Dawes, E. A., Microbiol. Rev. 54: 450-472, 1990). These PHA copolymers are synthesized by the same PHB synthase using the coenzyme A thioester derivatives of the C.sub.3 to C.sub.5 organic acids.
In addition to PHB and copolymers containing PHB, there is another general class of PHAs containing monomer units ranging between C.sub.6 and C.sub.12. Pseudomonas olevorans is the prototypical bacterium synthesizing PHAs containing medium-chain (D)-3-hydroxyacids when n-alkanes or n-alkanoic acids are provided in the growth media. The best studied of these PHAs is polyhydroxyoctanoate, which is accumulated when P. olevorans is grown in a medium containing octanoate (Huisman, G. W., de Leeuw, O., Eggink, G., and Witholt, B. Appl. Environ. Microbiol. 54: 2924, 1988). Furthermore, unique polymers possessing unsaturated or branched chain monomers, as well as possessing chloride or fluoride side groups, can be obtained by manipulation of the fermentation feedstock (Doi, Y. (ed), In: Microbial polyesters, Chpt. 3, VCH Publisher, New York (1990).
PHB is a stiff and relatively brittle thermoplastic (Doi, Y. (ed) In: Microbial Polyesters, Chpt. 6, VCH Publishers, New York, 1990; Holmes, P. A. In: Developments in crystalline polymers-2. Basset, D. C. (ed), 1-65, 1988). Incorporation of 3HV monomers into the polymer leads to a decrease in crystalinity and melting point compared to PHB, resulting in a decrease in stiffness and an increase in toughness of the polymer, making P(3HB-co-3HV) and other related copolymers more suitable for many commercial applications. It is also possible to blend various polymers and plasticizers to PHB in order to improve its physical characteristics (Holmes, P. A. In: Developments in crystalline polymers-2. Basset, D. C. (ed), 1-65, 1988). PHB has good UV resistance but generally poor resistance to acids and bases as well as organic solvents. PHB possesses good oxygen impermeability and is resistant to hydrolytic degradation in moist air. These properties makes PHB attractive as a source of plastic for a wide range of commodity products, such as household containers, bags and wrapping films. In contrast to PHB, long-chain PHAs are elastomers with a melting point ranging from 40.degree.-60.degree. C. (Gross, R. A., De Mello, C., Lenz, R. W., Brandl, H. and Fuller R. C., Macromolecules 22: 1106, 1989). The physical properties of these PHAs have yet to be fully characterized.
PHB and related copolymers are readily degraded in soil, sludge and sea water. For example, in soil at 30.degree. C., films of P(3HB-co-4HB) copolymer and PHB homopolymer are decomposed in two and ten weeks, respectively (Doi, Y. (ed) In: Microbial Polyesters, Chpt.1, VCH Publishers, New York, 1990). A number of bacteria and fungi were shown to be able to actively degrade these polymers (Dawes, E. A. and Senior, P. J., Adv. Microb. Physiol. 10:135-266 (1973). Extracellular PHB depolymerases and hydrolases have been isolated from several bacteria, including Alcaligenes feacalis. PHB can thus be degraded to monomeric 3HB units which can be used as a source of carbon for bacterial and fungal growth. Furthermore, PHB is very biocompatible, making it potentially attractive for medical applications such as suture filaments and drug carriers (Koosha, F., Muller, R. H. and Davis, S. S. In: Critical reviews in therapeutic drug carrier system, 6: 117-129, 1989). Degradation of PHB produces D-3-hydroxybutyric acid, a metabolite normally present in blood. The biodegradation of PHB is an important aspect of its usefulness as a plastic for commodity disposable products, as well as for specialized uses such as in agricultural mulches or medical implants.
P(3HB-co-3HV) copolymer synthesized by A. eutrophus is produced industrially by Imperial Chemical Industries and marketed under the trademark BIOPOL. Estimated cost based on a production of 500000 kg of polymer a year is approximately $15 per kg, in contrast to approximately $1 per kg for petroleum-derived commodity plastics such as polypropylene (Poole, R, Science 245: 1187-1189, 1989). Two major contributors to the cost of production are the carbon source added to the feedstock (eg sucrose, glucose, propionate) and harvesting of the polymer from the bacteria. A number of strategies are being explored to reduce the production cost (Poirier, Y., Dennis, D. E., Nawrath, C. and Somerville, C. Adv. Mater. 5: 30-36, 1993). For example, some bacteria are able to produce PHB when grown on cheap unrefined sugar sources such as molasses and corn syrup. Some strains of Pseudomonas and Rhodococcus are able to produce a number of PHA copolymers when grown on glucose, thus avoiding the addition of expensive substrates, like propionate, normally required for copolymer production. Genetic engineering of bacteria, including the use of E. coli synthesizing PHB, is also expected to have an impact on production cost of PHB. However, despite these potential improvements, it is generally agreed that due to the inherent costs associated with bacterial fermentation and downstream processing, the cost of PHA produced by bacteria will probably not be lower than approximately $3-5 per kg. It is unlikely that it will ever be possible to produce bacterial biomass at a cost comparable to that of producing biomass from higher plants. For example, potato can yield approximately 20000 kg of starch per hectare, with the potato tuber accumulating starch up to 80% of its dry weight (Martin, J. H., Leonard, W. H. and Stamp, D. L. (eds), Chapter 36, In: Principles of Field Crop Production, 898-932, Macmillan, New York, 1976). Starch is one of the lowest priced (approximately $0.2 /kg) and most abundant worldwide commodities. Similarly, oil producing crops, such as rapeseed, produce 1000 kg of oil per hectare with seed oil content up to 44% dry weight (Downey, R. K. and R obbelen, G., In: Oil crops of the world, R obbelen, G., Downey, R. K. and Ashri, A. (eds), Chpt. 16, McGraw-Hill, New York, 1989). In addition to be highly productive, plants have been shown to be very effective in producing a number of biologically active foreign proteins, such as antibodies (Hiatt, A., Cafferkey, R. and Bowdish, K., Nature 342: 76-78, 1989). There is growing interest in making use of the high productivity and flexibility of plants to produce a variety of organic materials, including proteins and various other polymers (Moffat, A. S., Science 256: 770-771, 1992).
Production of poly D-(-)-3-hydroxybutyrate, one member in the family of PHAs, has previously been demonstrated in the higher plant Arabidopsis thaliana (Poirier, Y., Dennis, E., Klomparens, K. and Somerville, C., Science 256: 520-523, 1992 and patent application Ser. No. 07/732,243). Of the three enzymes required to make PHB from acetyl-CoA, the 3-ketothiolase is endogenously present in plants. In the initial experiments, the genes from the bacterium Alcaligenes eutrophus encoding 3-ketothiolase (phbA), the acetoacetyl-CoA reductase (phbB) and the PHB synthase (phbC) were transferred and expressed in Arabidopsis under the transcriptional control of the constitutive CaMV 35S promoter (FIG. 1). In these experiments, the enzymes were targeted to the cytoplasm, because of the absence of organelle targeting signals on the gene products. Through appropriate genetic crosses, a hybrid plant was obtained which contained all of the enzymes required for PHB synthesis. Analysis of the chloroform-soluble compounds present in the hybrid plant by gas chromatography and mass spectrometry (GC-MS) revealed the presence of PHB. Between 20 to 100 .mu.g of PHB per gram fresh weight of plant material could be detected. Examination by electron microscopy of thin sections of plant tissues producing PHB revealed the presence of agglomerations of electron-lucent granules. These granules were very similar in size and appearance to the granules found in A. eutrophus and other bacteria accumulating PHB. Surprisingly, PHB granules were found in various compartments, namely the nucleus, vacuole and cytoplasm. No PHB granules could be detected in the mitochondria or chloroplast. The basis for this distribution of granules is unknown.
The demonstration of PHB production in genetically engineered Arabidopsis plants revealed several problems. One of the problems is the low yield of PHB. A second problem is the adverse effect of the expression of the phb genes on plant growth. Expression of high amounts of acetoacetyl-CoA reductase activity in transgenic plants caused a significant reduction in growth and seed production relative to wild type plants. For example, in a transgenic line expressing approximately 9 units of acetoacetyl-CoA reductase activity per mg of protein (one unit being defined as one .mu.mole of acetoacetyl-CoA reduced per min), the fresh weight of 22 day-old shoots was reduced to 19% of wild type (Poirier, Y., Dennis, D. E., Klomparens, K., Nawrath, C. and Somerville C., FEMS Microbiol. Lett., 103: 237-246, 1992). Seed production was reduced in approximately the same proportion. This phenotype could be the result of the diversion of a significant amount of acetyl-CoA and/or acetoacetyl-CoA away from essential biochemical pathways leading to a decrease in the production of compounds such as phytohormones, carotenoids, sterols, quinones, flavonoids, and lipids (FIG. 2). Alternatively, accumulation of a .beta.-hydroxybutyryl-CoA, or of a product derived from it, may be deleterious to plant cells. The fate of D-.beta.-hydroxybutyryl-CoA produced in transgenic plants is unknown. Expression of the PHB synthase, by itself, had no apparent effect on the growth or vigor of transgenic plants. However, hybrid plants containing both genes were more severely stunted in growth than plants containing only the acetoacetyl-CoA reductase activity. This could be due either to a more severe depletion of substrate from the mevalonate pathway or to a noxious effect of the PHB granules, particularly the granules being accumulated in the nucleus.